Stochastic sensing is based on the detection of individual binding events between analyte molecules and a single sensor element. Upon binding, a property of the sensor element is altered. This property or the effects of the changed property are measured.
In a simple example, the sensor element is a protein that is altered when it binds another molecule. The binding molecule to be detected is referred to as the analyte. The alteration of the sensor element that occurs upon binding is measured either directly or indirectly. In simple systems the alteration produces a simple signal, such as a difference in electrical current, force or fluorescence. Measurements of the signal indicate whether the analyte is bound and how long it remains bound. The frequency of occurrence of binding events is determined by the concentration of the analyte. The nature of the binding event is determined by the binding properties of the analyte, which determine, for example, the magnitude and duration of the resulting signal. Thus, a single sensor element to which multiple analytes may bind either directly may be used to determine which of those analytes are in a solution and the concentration of each particular analyte.
Although in simpler systems the sensor element has one binding site to which all analytes bind directly, it is possible for the sensor to have multiple binding sites, with different sites for different analytes. Additionally, a host or adaptor molecule may be used to facilitate binding of the analyte to the sensor element. The host molecule may merely facilitate the direct interaction of the analyte and sensor element, or it may serve as an adaptor that binds to both the analyte and the sensor element and allows connection of the two.
Stochastic sensing may be accomplished with various sensing elements, using various modes of detection. One simple model uses an ion channel protein pore embedded in a membrane between a cis chamber and a trans chamber. When the pore is fully open a large ion flux occurs (e.g. 108 ions/s) which constitutes an electrical current that may be monitored by single channel recording. When an analyte binds to the pore, ion flux is altered, usually by decreasing the flow of ions. This generates a current trace which shows conduction over time.
One particular pore that has been used in stochastic sensing is Staph alpha hemolysin (α(HL), which is actually an exotoxin secreted by Staphylococcus aureus. The monomeric 293 amino acid polypeptide can self-assemble on lipid bilayers, such as membranes, to form a heptameric pore. Alternatively, pre-formed pores may be inserted into a lipid bilayer. The pore is a mushroom-shaped structure in which the lower half of the stem forms a transmembrane channel. The interior of the pore is referred to as the “lumen” and may be accessible from outside the pore. By convention, when the pore is situated in a membrane, the side of the membrane on which the top of the mushroom shape is located is designated as the “cis” side of the membrane. The side of the membrane to which the stem portion leads is designated the “trans” side of the membrane. The pore essentially forms a hole in the membrane through which ions will flow if an electric potential is generated between the two chambers. (See FIG. 1.)
Stochastic sensing methods have been previously described in a number of publications, including U.S. Pat. No. 6,426,231 to Bayley et al. and a divisional application of that patent, U.S. patent application Ser. No. 10/180,792, filed Jun. 25, 2002. Protein pores for use in stochastic sensing and methods of using such pores have also been described in U.S. patent application Ser. No. 09/781,697 filed Feb. 12, 2001.
However, these previous manifestations of stochastic sensing have utilized non-covalent interactions between the analyte and the sensor element. There is considerable interest in the detection of reactive molecules including chemical warfare agents, pesticides, chemotherapeutic agents, and so on which will covalently bond to a sensor element. The reactivity of such molecules may be utilized to facilitate sensing and distinguish the reactive molecules from unreactive molecules.